tbuckley/cabot-search huggingface dataset:
P05.03.B METHYLOME PROFILING OF GLIOBLASTOMAS WITH FGFR3/TACC3 FUSION
Authors: A Picca, T Gareau, B Mohand Oumoussa, C Carpentier, A Dridi-Aloulou, A Di Stefano, K Mokhtari, F Bielle, M Sanson
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Glioblastomas (GBMs) with the FGFR3-TACC3 (F3T3) gene fusion have histological, molecular, metabolic, and clinicoradiological characteristics. They have a better prognosis compared to other GBMs. Recent findings suggest that methylome profiling could further stratify these patients identifying a subgroup (defined as “outlier” F3T3+ GBMs, or GBM-F3T3-O) with a methylation profile resembling gangliogliomas (GGs), low-grade histological appearance, and a better outcome. MATERIAL AND METHODS We performed DNA methylation profiling (Infinium MethylationEPIC Array v1.0) of 32 adult diffuse gliomas with FGFR3 gene fusions (30 FGFR3-TACC3, one FGFR3-ACLY, and one FGFR3-MYO18A), plus two paired samples of a diffuse glioma with two distinct contingents (one with positive FGFR3 staining and FGFR3-TACC3 gene fusion, the other with intense EGFR staining and EGFR gene amplification). All cases corresponded to “glioblastoma, IDH-wildtype” according to the WHO CNS5. Methylation data were submitted to the DFKZ brain tumor classifier (molecularneuropathology.org), versions 11b4 and 12.5 and to dimensionality reduction with t-distributed stochastic neighbor embedding (tSNE). Copy-number variation (CNV) estimation based on conumee package was obtained from DFKZ website. RESULTS With the brain tumor classifier v11b4, most cases (26/34, 76%) were classified as methylation class (MC) GBM (17/26 with a significant score ≥0.9). Out of them, 23/26 (88%) were significantly (score ≥0.5) classified in the “GBM, mesenchymal” (16/26, 62%) or in the “GBM, RTK II” methylation families (7/26, 27%). With the classifier v12.5, again most tumors were classified as “GBM, mesenchymal” (18/34, 53%) or “GBM, RTK II” (9/34, 26%), of which twelve (12/34, 35%) with a score ≥0.9. Two were classifed as GG (score 0.69 and 0.39), both having “GBM, mesenchymal” as a second hit (score 0.19 and 0.33, respectively). Five had GG as a second hit, although with low scores (range &amp;lt;0.01-0.25). After tSNE dimensionality reduction of our and literature cases, most tumors clustered together with other GBM, mesenchymal and GBM, RTK II cases. Only two cases fell within a distinct cluster corresponding to GBM-F3T3-O tumors from the literature. These included two histologically defined GBMs with a survival of 26 and 100 months. Recurrent inferred CNV included chromosome (chr) 7 gain (96%) and chr10 loss (85%), followed by chr20 gain (46%), chr22q loss (46%), and chr19 gain (33%). The presence of chr19 gain (alone or associated with chr20 gain) was associated with increased survival (p=0.01). CONCLUSION Glioblastomas with FGFR3 fusions (mostly F3T3) have an epigenomic profile close to other mesenchymal and RTK II GBMs. Methylation analysis identifies methylation (GBM-F3T3-O) and CNV (chr19 gain) profiles associated with better survival.
P11.47.B DNA METHYLATION-BASED SUBCLASS PREDICTS THE EFFECT OF THE EXTENT OF RESECTION ON SURVIVAL IN IDH-WILDTYPE GLIOBLASTOMA
Authors: L G Dankmeijer, L G Westerink, P C de Witt Hamer, P Wesseling, M C M Kouwenhoven, E Kooi, N Verburg
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND DNA methylation-based classification of diffuse glioma is increasingly used in daily practice. Yet, little is known regarding the prognostic and predictive value of DNA methylation-based subclasses of IDH-wildtype glioblastoma. A recent study found a predictive value for the effect of extent or resection (EOR) on survival for DNA methylation-based subclasses using the v11.4 classifier. We set out to validate these findings in an independent cohort and to test if these results are similar using the newest v12.5 classifier. MATERIAL AND METHODS A single-center retrospective cohort study of all patients with a glioblastoma, IDH-wildtype of whom DNA methylation was obtained. Patient and tumor characteristics, pre-operative Karnofsky performance status, and EOR (complete, subtotal, partial, biopsy) were retrieved. DNA methylation-based classification and MGMT promotor status were obtained using version 11.4 and 12.5 of the Heidelberg classifier. Progression-free survival (PFS) and overall survival (OS) were collected and validated using the national brain tumor registry. RESULTS Of 149 patients, the subtype was Mesenchymal in 57 (38.27%), RTK1 in 41 (27.52%), and RTK2 in 51 patients (34.23%). PFS and OS did not differ between the three subclasses, also not after pooling RTK1 and RTK2 into one Classical subclass. Complete or subtotal resection resulted in a longer OS than partial resection or biopsy in the entire cohort. This was similar for the pooled Classical subclass. In the Mesenchymal subclass, however, there was no survival advantage for complete or subtotal resection over partial resection or biopsy. These results were similar with the 11.4 and 12.5 classifier version. CONCLUSION In IDH-wildtype glioblastoma of the Mesenchymal subclass complete or subtotal resection does not result in a survival benefit. A less aggressive surgical strategy should be considered in these patients. Also, there is need for pre- or intraoperative assessment of the DNA methylation-based subclass of IDH-wildtype glioblastoma.
P06.16.A FRACTIONATED IRRADIATION ACTIVATES GLIOMA IMMUNE MICROENVIRONMENT AND BOOSTS ANTIGEN-SPECIFIC T CELL RESPONSE IN MALIGNANT GLIOMAS
Authors: N Elmadany, J Michel, J Sonner, S Ovchinnikova, K Sanghvi, I Bernhardt, X Zhang, K Jähne, M Ott, A Sati, S Anders, M Breckwoldt, M Platten, K Sahm
Journal: Neuro-Oncology (2023)

Abstract Several ongoing clinical trials employ vaccine, immune checkpoint inhibitors and/or adoptive T cell therapy to treat glioblastoma. However, the impact of irradiation as part of the standard of care on the immune microenvironment and response to immunotherapy is ill-defined. In the present study, we analyzed the post-irradiation changes in the GBM immune microenvironment, and further elucidated the impact of fractionated tumor irradiation (FIR) on the efficacy of T cell-mediated immunotherapies in experimental gliomas. FIR boosted the response of orthotopically-implanted GL261 tumors expressing the tumor-associated antigen glycoprotein (gp)100 to a gp100 vaccine. Furthermore, FIR enhanced the infiltration of GL261-gp100 gliomas by adoptively-transferred gp100 T cell receptor-transgenic T cells, which also displayed a more activated and less exhausted phenotype post-irradiation. Single cell transcriptomic studies of tumor-infiltrating CD45+ leukocytes revealed that FIR promotes the expression of leukocyte-endothelial adhesion molecules in T cells, and transcripts encoding proinflammatory M1-like genes in tumor-associated microglia and macrophages. Collectively, our preclinical findings support the use of FIR with to increase response to T cell-based immunotherapies.
P12.03.A MULTIMODAL ANALYSIS OF GLIOBLASTOMA REVEALS THE POTENTIAL OF CXCR7-CXCL12 IN GBM CELLS SENSITIZES GBM TO IMMUNE CHECKPOINT BLOCKADE THERAPY
Authors: C Liu, C Chien, W Yang, J Chuang, K Chang
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Glioblastoma (GBM), a WHO Grade IV glioma, is one of the most lethal brain tumors. In spite of the standard of care and currently developed advanced treatment, the prognosis of GBM patients is still poor. In addition to the understanding of GBM cells, the microenvironment plays a critical role in regulating GBM progression. Also, more and more information indicates the interaction of GBM-associated macrophages (GAMs) and GBM cells facilitates GBM progression via sculpturing immunosurveillance, cancer stemness, and cancer resistance. And thus, we hypothesized that GBM cells sculptured the microenvironment via macrophage alternation of which interventions may potentialize immune checkpoint blockade therapy. MATERIAL AND METHODS The bulk RNA sequencing (seq.) of the paired primary-recurrent GBM specimens, single-cell RNA seq., in vitro (including mRNA, protein, and cell behavior tests), and in vivo validation, were integrated to explore our hypothesis. RESULTS The bulk RNA seq. of the paired primary-recurrent GBM specimens showed recurrent GBM enhanced the neuroinflammation pathway. Among the secretory components, CXCL12, also known as stromal cell-derived factor 1 was accumulated during GBM progression and upregulated in temozolomide (TMZ)-resistant GBM cells compared with parental GBM cell lines. In addition, the newly CXCL12 receptor CXCR7 may act as a scavenger to breakdown CXCL12-CXCR4 positive feedback in GBM resistance. Moreover, the bulk RNA sequencing seq. showed that recurrent GBM reduced CXCR7 expression. Single-cell RNA seq. of GBM patients’ specimens suggested that CXCR7 was majorly expressed in GBM cells than in normal neural and immune cells. Developing TMZ resistance in GBM cell lines also down-regulated CXCR7 expression. Furthermore, silencing CXCR7 attenuated TMZ cytotoxicity while silencing CXCR7 and CXCL12 simultaneously reversed the effects. In contrast, combining CXCR7 agonists and TMZ enhanced TMZ cytotoxicity Otherwise, the GBM-associated CXCL12 activated M0 macrophages into GAMs by facilitating macrophage proliferation and inducing pro-tumor factors, including IL-1β and its receptors, IL-6, MMP9, and immune checkpoint PD-L1. Mechanistically, GBM-associated CXCL12 upregulated PD-L1 in GAMs via NF-κB. Also, in the orthotopic GBM mouse model, CXCR7 knockdown in GBM cells led to poor survival and increased PD-L1 expression in GAMs. Finally, we combined CXCR7 agonist and anti-PD-L1 to show prolonged survival, in contrast, anti-PD-L1 did not show this effect. CONCLUSION In accordance with the multimodal analysis, GBM regulated the accumulation of CXCL12 via silencing CXCR7 in GBM cells. The CXCR7-CXCL12 axis confers the immunosuppression by up-regulating PD-L1 in GAMs. Our study suggested that CXCR7 activation treatment alone or combined with an immune checkpoint inhibitor may be a potential strategy for the treatment of GBM.
P08.05.A A LONGITUDINAL CASE STUDY OF PATIENTS' EXPERIENCES IN THE INITIAL PHASE OF A BRAIN CANCER PATHWAY - AIMING TO IMPROVE PATIENT CARE
Authors: T Vedelø, J Sørensen, E Hall, C Delmar
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND A brain cancer diagnosis is life-transforming and involves accelerated diagnostics and treatment and care involving multiple settings and healthcare professionals. Patients are confronted with an uncurable cancer disease but also face progressive neurological and cognitive deficits over time, adding to the uncertain and stressful situation. Knowledge of patients' experiences during their hospital stay in the initial diagnostic phase is sparse. This study aimed to explore patients' experiences at the hospital during the diagnostic workup, the brain surgery, and the post-operative period until discharge and to describe what matters to patients when diagnosed with a malignant brain tumour. MATERIAL AND METHODS We used a descriptive longitudinal single-case study design to produce in-depth, context-dependent knowledge and capture real-life situations in patients' day-to-day care experiences from admission to discharge across three different departments throughout the initial diagnostic phase. To explore patients' views directly in the actual situations unfolding in practice, we followed one patient at a time and did 180 hours of field observation and 16 semi-structured interviews. We analyzed field notes and interviews by applying systematic text condensation. RESULTS An overarching theme, "A fast transition", was identified, describing the patients' experiences in the two central themes; "From feeling fine to an unknown journey" and "Patient involvement determined by system factors". Within the two themes, six subthemes emerged from the data: Overwhelming uncertainty and fear, Putting life on hold, An unfamiliar dependency and Being unprepared and side-lined, The significance of close relatives, and Managing care needs and demands. CONCLUSION The results add to understanding patients' experiences during the initial diagnostic phase. While part of the findings are a fact of life for patients diagnosed with a brain tumour, others point to specific areas for improvement. Small changes in care delivery can enhance patient involvement by proactively informing patients about their care plan, ensuring the presence of relatives during information giving and identifying patients' care needs and preferences. Establishing a patient and public involvement group in 2023 will provide more knowledge gaps for future research.
P02.16.B COMPENSATORY CROSS-TALK BETWEEN AUTOPHAGY AND GLYCOLYSIS REGULATES SENESCENCE AND STEMNESS IN HETEROGENEOUS GLIOBLASTOMA TUMOR SUBPOPULATIONS
Authors: H Senthil, E Martell, H Kuzmychova, E Kaul, C Venugopal, S K Singh, T Sharif
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Despite tremendous research efforts, the successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and metabolic plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Hence, a deeper understanding of the cellular adaptations in response to metabolic interventions is urgently required to develop more effective therapeutic options against deadly glioblastoma (GBM) brain tumors. MATERIAL AND METHODS Using clinically-relevant patient-derived GBM models, we discovered a cross-talk between glycolysis, autophagy, and senescence. Protein and mRNA expression of glycolytic enzymes and stemness markers were measured using western blot and bioinformatic analysis, respectively. We were able to measure senescence with the use of β-galactosidase staining and upregulation of cell cycle regulators, such as p21/CDKN1A and p16/CDKN2A. Autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis was used to determine autophagy induction. RESULTS We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Moreover, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or triggering apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. CONCLUSION These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress. This work was supported by the Cancer Research Society (CRS).
P10.01.A A PATIENT-DERIVED<i>EX VIVO</i> ORGANOID MODEL ALLOWS EVALUATION OF TUMOR TREATING FIELDS (TTFIELDS) EFFECTS ON GLIOBLASTOMA, ANAPLASTIC EPENDYMOMA AND OLIGODENDROGLIOMA SAMPLES
Authors: V Nickl, E Schulz, E Salvador, L Trautmann, L Diener, A F Kessler, C M Monoranu, R Ernestus, M Löhr, C Hagemann
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND The use of TTFields, which are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), has shown its clinical efficacy in the treatment of newly diagnosed glioblastoma (GBM) patients at 200 kHz. Nevertheless, more knowledge is required for the prediction of individual tumors’ responsiveness to treatment. Recently, we developed patient-derived three-dimensional GBM tissue culture models to screen patient tumors for TTFields treatment susceptibility. Here, we extended these models to brain tumors beyond GBM, namely anaplastic ependymoma (AE) and oligodendroglioma. We cultured tumor-organoids as microtumors on murine organotypic hippocampal slice cultures (OHSCs) and subjected them to TTFields at 200 kHz. MATERIAL AND METHODS We prepared OHSCs by slicing the brains of mice 5-8 days postpartum to a thickness of 350 µm using a vibratome and culturing them for 14 days. We obtained organoids from freshly resected tumor tissue from three GBM, one AE, and one oligodendroglioma patient, each. The organoids were cultured for 14 days as well and then stained with carboxyfluorescein succinimidyl ester. Next, we placed the organoids onto the OHSCs and administered TTFields at 200 kHz and 1.5 V/cm for 72 h using the inovitro™ laboratory research system. We quantified microtumor growth by fluorescence imaging. RESULTS The three tumor entities, in particular organoids from GBM, AE and oligodendroglioma, grew as microtumors after being placed onto OHSCs. TTFields treatment at 200 kHz led to significantly decreased microtumor size of the three analysed GBM samples as well as the AE sample. However, the oligodendroglioma microtumor size was not significantly reduced. CONCLUSION Based on clinical experience, we expected that TTFields lead to a reduction in microtumor size. However, to our knowledge, we were the first to apply TTFields to patient-derived AE and oligodendroglioma samples. The lack of TTFields-effects on the latter might be due to different inter-patient sensitivity or suboptimal TTFields frequency. Nevertheless, our work is proof of principle that an organoid-based patient-derived 3D tissue culture model could be used to evaluate effects of TTFields on different brain tumor entities.
P04.07.B CHARACTERIZATION OF ARHGAP36/MYC-INDUCED GROUP 3 MEDULLOBLASTOMA
Authors: M Gianesello, C Ballabio, A Badaloni, D Bonanomi, L Tiberi
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Medulloblastoma (MB) is one of the most common pediatric brain cancers of the central nervous system. It is stratified into 4 molecular subtypes (WNT, SHH, group 3, and group 4), which differ from one to the other for various features, such as cellular morphology, age of onset, set of mutations, and percentage of survival. A common point between the four MB subgroups is the absence of treatments for this disease. The current gold standard treatment has deleterious side effects for the patients, which spans from growth defects to low hormone levels and problems with learning. Analyzing transcriptomic data of group 3 MB patients we found ARHGAP36 as a possible gene involved in tumor formation or propagation. Rho GTPase-Activating Protein 36 (ARHGAP36) is a gene that plays a role in regulating PKA activity. Specifically, the ARHGAP36 protein can inhibit PKA function through two distinct mechanisms. Firstly, it acts as a pseudosubstrate for the catalytic subunit of the PKA protein. Secondly, it can drive protein degradation via a lysosome-mediated process. MATERIAL AND METHODS We took advantage of the Piggybac/transposon technology to induce a strong and stable overexpression of our genes of interest to generate two different models, one in-vivo and one in-vitro with induced pluripotent stem cells-derived human cerebellar organoids. We also exploited the cre/LoxP system in transgenic mice to overexpress ARHGAP36 under the control of specific promoters. The analysis was conducted using standard techniques such as RT-qPCR and Western Blot, together with immunofluorescence of the cerebellar section and live imaging of the modified cerebellar organoids. The gene expression profile of the tumors obtained in mice was analyzed by RNA sequencing and compared to known group 3 MB mouse models. RESULTS We analyzed RNA-sequencing data from medulloblastoma patients and found that high expression of ARHGAP36 correlated with poor prognosis in patients. Through PKA inhibition, ARHAP36 induces ectopic activation of SHH signaling and cell proliferation leading to abnormal development of the cerebellum in mouse embryos. Notably, despite its inductive activity on the SHH pathway, ARHGAP36 expression is higher in group 3 and group 4 MB, rather than in the SHH subgroup. In this study, we demonstrate that intracranial transfection of P0 mouse pups with plasmids overexpressing MYC and ARHGAP36 can generate group 3 medulloblastoma in-vivo. We studied and then confirmed the involvement of the binding with PKA as a tumorigenic mechanism in the MYC/ARHGAP36 co-overexpression using several mutated forms of the gene. We studied the obtained tumors for the presence of group 3 MB immunophenotypical markers, as well as their gene expression profile by RNA-seq. CONCLUSION These findings can become of key interest as possible druggable targets to efficiently treat group 3 medulloblastoma patients in the future.
P10.09.A TARGETING NITROSATIVE STRESS TO PREVENT NEUROBLASTOMA MALIGNANCY: INSIGHTS INTO THE MTOR SIGNALING PATHWAY
Authors: S K Ojha, M K Tripathi, W Hamoudi, I Khaliulin, H Amal
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Neuroblastoma (NB) represents the most common pediatric tumor and its mechanisms remain elusive. Nitric oxide (NO) is one of the essential regulators of carcinogenesis of different tumors including NB. The mechanistic target of the rapamycin (mTOR) signaling pathway is another important regulator of tumorigenesis. In this study, we investigated the possibility to attenuate NB malignancy by inhibiting nitrosative stress and examined the interplay between NOS and mTOR signalling. MATERIAL AND METHODS NOD.CB17-Prkdc-scid/NCrHsd mouse were used for xenograft subcutaneous tumor model to evaluate the ability of HU-53 treatment regimens. Tumor weight and volume were measured during the study. The experiments were also carried out on the human-derived SH-SY5Y cells. Nitrite content was measured biochemically by griess assay. Immunoblotting and immunofluorescence assessed nitrosative stress and its effect on mTOR signaling. Genetic knockdown of nNOS was done in SH-SY5Y with siRNA to check effect of NO on neuroblastoma growth. RESULTS Treatment with HU-53 resulted in a significant decrease in tumor weight (p &amp;lt; 0.0005) and volume (p &amp;lt; 0.0002) as compared to mice treated with vehicle. Furthermore, we observed that the HU-53 treatment resulted in decreased levels of nitric oxide and nitrosative stress as confirmed by checking the level of nitrite content and 3-Nitrotyrosine which are important mediators of neuroblastoma growth. Genetic nNOS knock-down also prevented cell proliferation, reduced nitrosative stress (p &amp;lt; 0.0002), and the level of synaptophysin, the marker of NB carcinogenesis. Interestingly, NO inhibition elevated the levels of the core negative regulator of mTOR signaling, TSC2 and decreased the phosphorylation of the components of the mTOR signaling pathway, AKT, mTOR, and RPS6, indicating the reduced activity of this pathway. Thus, these results show inhibition of NO production help in reduced activation of mTOR pathway and prevent tumor growth. CONCLUSION Our results indicate that nitric oxide inhibition prevents neuroblastoma tumor growth and suggest that the NO-mTOR axis could serve as a novel potential target for NB treatment.